Dopamine (DA) is a major neurotransmitter with diverse physiological impact, and is required for movement, working memory, and reward. Following evoked release, DA extracellular half-life is determined by presynaptic reuptake, mediated by the SLC6 plasma membrane DA transporter (DAT). DAT is the primary target for addictive and therapeutic psychostimulants, such as amphetamine, cocaine and methylphenidate (Ritalin), which potently inhibit DA uptake, sustain DA signaling and impact DA-dependent behaviors. DAT coding variants are implicated in a variety of neuropsychiatric disorders, and transgenic mouse studies clearly demonstrate that DAergic signaling and behaviors, as well as psychostimulant efficacy, are highly sensitive to the level of DAT expression. DAT is not static at the plasma membrane, but is subject to robust constitutive endocytic recycling. Moreover, direct protein kinase C (PKC) activation rapidly accelerates DAT internalization and decreases DAT surface availability and function. Efforts over nearly two decades have elucidated many of the mechanisms underlying basal and PKC-regulated DAT trafficking. However, it remains unclear how DAT is regulated in bona fide presynaptic DA terminals, whether DAT regulation is region-dependent, and whether DAT trafficking impacts DA signaling. Moreover, it is unknown whether DAT trafficking mechanisms identified in cell line studies are physiologically relevant. The major goals of the proposed studies are to determine the presynaptic mechanisms that regulate DAT surface expression, and test whether DAT trafficking has physiological impact on DA release and clearance. Specifically, we aim to test 1) whether presynaptic Gq-coupled receptor activation impacts DAT surface expression and function in a region-dependent manner, 2) whether retrograde signaling within the striatum regulates presynaptic DAT surface expression and function, and 3) whether DAT trafficking dysfunction impacts DA signaling. These hypotheses stem from strong preliminary data that demonstrate region-specific, Gq-mediated, biphasic DAT trafficking in striatal DAergic terminals, and putative roles for the Gq-coupled M5 and Gi-couple NOPR receptors in presynaptic DAT trafficking. These provocative findings suggest that multiple mechanisms converge on DAT within the striatum to regulate DAT surface availability and function. To pursue this investigative line, we will leverage a variety of state-of-the-art mouse genetic tools, including a newly developed viral approach to conditionally and inducibly achieve shRNA-mediated gene knockdown in DA neurons, as well as chemogenetic and optogenetic strategies. Ex vivo striatal slice studies will measure changes in DAT surface expression in response to presynaptic receptor activation, and fast-scan cyclic voltammetry will measure DA release and DAT-mediated clearance, in parallel. Conditional transgene expression, gene ablation and shRNA-mediated knockdown strategies will be used to define the cell autonomous mechanisms that impact DAT trafficking within mouse striatum. The information gleaned from these studies will provide a clearer understanding of DAT surface dynamics in native presynaptic terminals, and the impact that DAT trafficking imposes on DA signaling in the striatum. Moreover, we anticipate that our findings will greatly impact future strategies aimed at treating DA-related disorders, in which manipulating DAT function could provide therapeutic benefit.